The first thing to be aware of is and I quote from manual: "Due to light-induced auto-oxidation, DCFH-DA solutions at any concentration must be protected from light." So you need to be aware of any potential spectral overlap of your illumination and DCFH-DA absorbance. There is a possibility that this method is incompatible with PDT, however I think it might work with a careful selection of wavelengths. The way I would do it is as follows:

Seed the cells ~ 15000 and grow overnight. Incubate with PS for as long as required. For the last 30-60 min of incubation, remove media and replace with OPTIMEM (or -FBS) containing both PS and DCFH-DA. Remove wash replace media (complete) and irradiate at wavelength sufficiently higher than the DCFH-DA absorbance (I would go >600nm). Read DCFH-DA at excitation 490 and emmission 510-570 nm. Make sure that your PS does not absorb-emit at these wavelengths.

Thank you for your answer.

I must irradiate the photosensitizer at 435 nm, or else I will not manage to excite it...

What do you think about the concentration of DCFH-DA? Is 0.1mM too low?

I have also red that redox active metals may oxide DCFH-DA. I have been using PBS with Mg and Ca when I wash. I am not sure about they being redox active or not, though.

Hi Cathrine, I think your biggest problem is the irradiation wavelength. I haven't looked at the DCFH-DA abs spectrum but I presume that 490 is the maximum and since I presume you are using lamp and filter there is bound to be considerable overlap. 0.1 mM is what is recommended but you can double that... Still, I don't think this is your problem and nor transition metals. Are you after a particular ROS?

I am not after a particular ROS. Just general ROS generation from the photodynamic therapy process.

The strange thing is that the assay have worked well on an other cell line before. Perhaps my DCFH-DA is starting to get old and has already auto-oxided...

With the same sensitizer? Could be!... and irradiation does not particularly help.

With another sensitizer, but a sensitizer that was excited with the same wave length.

Here is the spectrum for the substrate:

http://probes.invitrogen.com/media/spectra/368ph9.jpg

The absorption is lying very near the excitation light I apply, but should be okay.

and your light source profile? How broad is that? One thing to do is try your DCFH-DA with the other photosensitizer that works. If it doesn't work now it may mean your DCFH is gone off. If it still works though....

Have you looked at the cells in a normal fluoresence microscope ? Do they appear stained ?

Are you taking the reading from top or bottom ? Sometimes it makes a difference

What about your positive controls ? Have you included them in the experiment?

Its better to have autofluoresence from each well before adding the fluorophore and used as a blank as every cell has variation in the no. of cells. This might affect results.

I haven't looked at them in a fluorescence microscope.

I take the reading from top.

I have not included a positive control this time. I did it when I studied my other sensitizer, and then it worked fine.

Now I started a new experiment where i doubled the DCFH-DA dose 10 times.

I feel you should include a positive control (say like hydrogen peroxide to keep it simple and easy) with this experiment as well and take your readings at different time points because its possible that the dye reaches saturation and then the fluoresence stops. and bottom reading is better in this case may.. you can try this.. and do not cover your plates with foil or a dark cover even if you take the reading from bottom. I feel 10 micro molar dcfh-da is sufficient but it may vary from cell to cell and you need to optimize the assay for each cell.

Thank you for your answers :)